Fibrillar collagens form the largest protein structures found in complex organisms (Piez, 1984, In: Extracellular Matrix Biochemistry, Piez et al., Eds., Elsevier, N.Y., pp. 1-40; Prockop et al., 1995, Ann. Rev. Biochem. 64:403-434). The most abundant collagen fibrils consist almost entirely of a single monomer of type I collagen. The structure of the monomer was established several decades ago, but the precise pattern of packing of the monomer into fibrils has not been defined and remains controversial (Smith, 1968, Nature 219:157-158; Hulmes et al., 1979, Nature 282:878-880; Holmes et al., 1979, Biochem. Biophys. Res. Commun. 87:993-999; Piez et al., 1981, Biosci. Rep. 1:801-810; Hulmes et al., 1981, Proc. Natl. Acad. Sci. USA 78:3567-3571; Brodsky et al., 1982, Methods Enzymol. 82:127-173; Woodhead-Galloway, 1984, In: Connective Tissue Matrix, Hukins, Ed., Verlag Chemie, Basel, pp. 133-160; Hulmes et al., 1985, J. Mol. Biol. 184:473-477; Ward et al., 1986, J. Mol. Biol. 190:107-112; Galloway, 1985, In: Biology of Invertebrate and Lower Vertebrate Collagens, Bairoti et al., Eds., Plenum Press, New York, pp. 73-82; Chapman, 1989, Biopolymers 28:1367-1382; Jones et al., 1991, J. Mol. Biol. 218:209-219).
Type I collagen is similar to other fibrillar collagen in that it is first synthesized as a soluble procollagen containing N-propeptides and C-propeptides (Prockop et al., 1995, Ann. Rev. Biochem. 64:403-434). The propeptides are cleaved by specific N- and C-proteinases and the monomers then spontaneously assemble into characteristic fibrils. The two xcex11(I) chains and one xcex12(I) chains of a monomer of type I collagen are primarily comprised of about 338 repeating tripeptide sequences of -Gly-Xxx-Yyy- in which -Xxx- is frequently proline and -Yyy- is frequently hydroxyproline. The ends of the xcex11(I) and xcex12(I) chains consist of short telopeptides of about 11 to 25 amino acids per chain. The distribution of hydroxyproline and charged residues in the -Xxx- and -Yyy- positions in the triple-helical domain define 4.4 repeats or 4.4 D-periods of about 234 amino acids each. In longitudinal sections, the monomers are arranged in fibrils in a head-to-head-to-tail orientation with a gap of about 0.6 D-periods and, therefore, repeat of 5 D-periods. The continuity of the fibrils is maintained by many of the monomers being staggered by 1, 2, 3, or 4 D-periods relative to the nearest neighbor so as to generate gap and overlap regions. However, there are conflicting data from electron microscopy and X-ray analysis about the lateral packing of the monomers. One view is that the monomers are laterally packed in a tilted quasi-hexagonal lattice (Hulmes et al., 1979, Nature 282:878-880; Jones et al., 1991, J. Mol. Biol. 218:209-219). A related view is that the fibrils consist of xe2x80x9ccompressedxe2x80x9d microfibrils that are comprised of monomers coiled into a rope-like pentameric structure (Smith, 1968, Nature 219:157-158; Piez et al., 1981, Biosci. Rep. 1:801-810). Still another view is that the lateral packing of the collagen in many fibrils is either liquid-like or a biological equivalent of a liquid crystal Galloway, 1985, In: Biology of Invertebrate and Lower Vertebrate Collagens, Bairoti et al., Eds., Plenum Press, New York, pp. 73-82; Chapman, 1989, Biopolymers 28:1367-1382).
One experimental approach to defining the lateral packing of the monomers was to observe the initial assembly of monomers into fibrils. Early experiments (Piez, 1984, In: Extracellular Matrix Biochemistry, Piez et al., Eds., Elsevier, N.Y., pp. 1-40; Ward et al., 1986, J. Mol. Biol. 190:107-112; Veis et al., 1988, In: Collagen: Biochemistry, Vol. 1, Nimni, Ed., CRC Press, Boca Raton, Fla., pp. 113-138) on the re-assembly of fibrils from collagen extracted from tissues with acidic buffers suggested that the first structures formed were linear strands of monomers bound by 0.4 D-period overlaps (4 D staggers). Other observations with extracted collagens suggested the initial stages involved assembly of structures similar -to pentarneric microfibrils (Piez, 1984, In: Extracellular Matrix Biochemistry, Piez et al., Eds., Elsevier, N.Y., pp. 1-40; Veis et al., 1988, In: Collagen: Biochemistry, Vol. 1, Nimni, Ed., CRC Press, Boca Raton, Fla., pp. 113-138; Gelman et al., 1980, J. Biol. Chem. 155:8098-8102). Subsequently, a system was developed for studying assembly of type I collagen fibrils de novo by enzymic cleavage of a purified soluble precursor of procollagen under physiological conditions (Miyahara et al., 1982, J. Biol. Chem. 257:8442-8448; Kadler et al., 1987, J. Biol. Chem. 262:15696-15701; Kadler et al., 1990, Ann. N.Y. Acad. Sci. 580:214-224; Kadler et al., 1990, Biochem. J. 268:339-343). Because thick fibrils were generated in the system, it was possible to use dark-field light microscopy to follow the growth of the fibrils through intermediate stages (Kadler et al., 1990, Biochem. J. 268:339-343). The first fibrils detected had a blunt end and a pointed tip or end. Initial growth of the fibrils were exclusively from the pointed or a-tip. Later, b-tips appeared on the blunt ends of the fibrils and the fibrils grew from both directions. Scanning transmission electron microscopy indicated that both the a-tips and b-tips were near paraboloidal in shape (Holmes et al., 1992, Proc. Natl. Acad. Sci. USA 89:9855-9859 Silver et al., 1992, Proc. Natl. Acad. Sci. 89:9860-9864). Also, it appeared that the monomers are oriented with their N-termini directed toward the tips. Subsequent experiments in the same system with type II collagen suggested that the fibrils also grew from pointed tips. However, the monomers were oriented with a C-termini directed toward the tips (Fertala et al., 1996, J. Biol. Chem. 271:14864-14869). Three different models were proposed to explain the growth of fibrils from near-paraboloidal tips. One model (Silver et al., 1992, Proc. Natl. Acad. Sci. 89:9860-9864) was based on the assumption that the initial core of the fibril was a pentameric microfibril and that the fibril grew by addition of monomers in a helical pattern. Simulations of the model suggested that as little as two specific binding steps were required first for assembly of the microfibrillar core and then a structural nucleus with about the same diameter as the final fibril. After assembly of the structural nucleus, the fibril grew from the paraboloidal tip by addition of monomers through only one of the two binding steps. A second and related model (Hulmes et al., 1995, Biophys. J. 68:1661-1670; Hulmes et al., 1989, J. Mol. Biol. 210:337-345) suggested that assembly began with formation of an undefined inner core and then monomers were added in spiral strands to generate the near-paraboloidal tips. The second model had the advantage that it more readily than the first model accounted for X-ray diffraction data that indicated that some fraction of monomers in fibrils were laterally packed in a tilted quasi-hexagonal lattice (Hulmes et al., 1979, Nature 282:878-880; Galloway, 1985, In: Biology of Invertebrate and Lower Vertebrate Collagens, Bairoti et al., Eds., Plenum Press, New York, pp. 73-82). In contrast to the first two models, a third model (Parkinson et al., 1994, Physical Rev. E. 50:2963-2966) was developed in which monomers were assembled by a process involving only aggregated limited diffusion. The third model, therefore, assumed that the assembly of monomers into fibrils was similar to processes such as electrochemical depositions or perhaps formation of snowflakes, and that the process did not require the presence of specific binding sites on the monomers.
The invention relates to type I collagen assembly-inhibiting peptides. These peptides inhibit assembly of human type I collagen, for example, in an in vitro collagen self-assembly assay. In one embodiment, the peptide is selected from the group consisting of a type I collagen xcex11 N-telopeptide, a type I collagen xcex11 C-telopeptide, a type I collagen xcex12 C-telopeptide, a type I collagen xcex12 C-telopeptide derivative. By way of example, the peptide may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4-11, and 13-23, preferably from the group consisting of SEQ ID NOs: 2 and 4-11, and more preferably from the group consisting of SEQ ID NOs: 4-8 and 11. In another embodiment, the peptide is a peptidomimetic of a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4-11, and 13-23.
The invention also relates to a method of identifying a type I collagen assembly-inhibiting peptide. This method comprises immobilizing a collagen species on a support. The collagen species is selected from the group consisting of human type I collagen and a human type I collagen peptide. The support is contacted with a labeled peptide in the presence or absence of an unlabeled test peptide. The labeled peptide is known to inhibit collagen self-assembly. The amount of labeled peptide bound to the support is assessed, and a lower amount of labeled peptide bound to the support in the presence of the test peptide compared with the level of binding of the labeled peptide to the support in the absence of the test peptide is an indication that the test peptide is a type I collagen assembly-inhibiting peptide.
The invention further relates to a method of identifying a type I collagen assembly-inhibiting peptide. This method comprises immobilizing a collagen species on a support. Again, the collagen species is selected from the group consisting of human type I collagen and a human type I collagen peptide. The support is contacted with at least one peptide-bearing particle which comprises a test peptide. It is then assessed whether the peptide-bearing particle binds with the support. Binding of the peptide-bearing particle to the support is an indication that the test peptide is a type I collagen assembly-inhibiting peptide. In one embodiment of this method, the human type I collagen peptide has the amino acid sequence of amino acid residues 776 through 796, inclusive, of the xcex11 chain of human type I collagen. In another embodiment, the peptide-bearing particle is a phage or a phage display peptide library. In a variation of this method, the support is rinsed prior to assessing whether the peptide-bearing particle binds with the support. In one embodiment of this method assessing whether the peptide-bearing particle binds with the support comprises contacting the support with a collagen un-binding eluent and detecting the presence of the peptide-bearing particle in the eluent. By way of example, the collagen un-binding eluent may be selected from the group consisting of Tris-glycine buffer at a pH of about 2.2, a suspension comprising human type I collagen, and a suspension comprising a peptide having the amino acid sequence of amino acid residues 776 through 796, inclusive, of the xcex11 chain of human type I collagen. In one embodiment of this method, the peptide-bearing particle is a phage, and detecting the presence of the phage comprises detecting the ability of the phage to lyse cultured cells. In another variation of this method, the peptide-bearing particle in the eluent is contacted with a second support, wherein the second support has the same or a different collagen species immobilized thereon.
The invention also relates to a method of inhibiting type I collagen self-assembly comprising contacting the collagen with a type I collagen assembly-inhibiting peptide. In one embodiment, the collagen is in a human.
The invention also relates to use of a type I collagen assembly-inhibiting peptide for preparation of a medicament for inhibiting type I collagen self-assembly in an animal, preferably in a human.
The invention also relates to a chromatographic medium comprising a support having a type I collagen assembly-inhibiting peptide immobilized thereon.
The invention further relates to a method of purifying human type I collagen. This method comprises contacting a relatively impure suspension comprising human type I collagen with a support having a type I collagen assembly-inhibiting peptide immobilized thereon and separating the support from the suspension to yield relatively purified human type I collagen.